The most conspicuous and important processes related to post-harvest quality of climacteric fruit are the changes in texture, color, taste, and aroma which occur during ripening. Because of the critical relationship that deleterious changes in texture have to quality and post-harvest shelf-life, emphasis has been placed on studying the mechanisms involved in the loss of firmness that occurs-during tomato fruit ripening. Although fruit softening may involve changes in turgor pressure, anatomical characteristics and cell wall integrity, it is generally assumed that cell wall disassembly leading to a loss of wall integrity is a critical feature. The most apparent changes, in terms of composition and size, occur in the pectic fraction of the cell wall (see references in Seymour and Gross, 1996).
Changes known to occur in the pectic fraction of the cell wall during fruit ripening include increased solubility, depolymerization, de-esterification and a significant net loss of neutral sugar containing side chains (Huber, 1983; Fischer and Bennett, 1991; Seymour and Gross, 1996). The best characterized pectin-modifying enzymes are polygalacturonase (endo-α1→4-D-galacturonan hydrolase; E.C. 3.2.1.15; PG) and pectin methylesterase (E.C. 3.1.1.11; PME). Although PG and PME are relatively abundant and have substantial activity during tomato fruit ripening, softening still occurs, albeit with a slight delay, in fruit where PG (Smith et al. 1988, 1990) or PME (Tieman et al. 1992; Hall et al. 1993) gene expression and enzyme activity was significantly down-regulated in transgenic plants. Moreover, over-expression of PG in non-ripening mutant rin tomato fruit did not result in softening even though depolymerization and solubilization of pectin was evident (Giovannoni et al., 1989).
Among the other known pectin modifications that occur during fruit development, one of the best characterized is the significant net loss of galactosyl residues which occurs in the cell walls of many ripening fruit (Gross and Sams, 1984; Seymour and Gross, 1996). Although some loss of galactosyl residues could result indirectly from the action of PG, β-galactosidase (exo-β(1→4)-D-galactopyranoside; E.C. 3.2.1.23) is the only enzyme identified in higher plants capable of directly cleaving β(1→4)galactan bonds, and probably plays a role in galactan sidechain loss (DeVeau et al., 1993; Carey et al., 1995; Carrington and Pressey, 1996). No endo-acting galactanase has yet been identified in higher plants. The view that β-galactosidase is active in releasing galactosyl residues from the cell wall during ripening is supported by the dramatic increase in free galactose, a product of β-galactosidase activity (Gross, 1984) and a concomitant increase in activity of a particular enzyme, designated β-galactosidase II, in tomatoes during ripening (Carey et al., 1995). β-galactosidase activity is thought to be important in cell wall metabolism (Carey et al., 1995). β-Galactosidases are generally assayed using artificial substrates such as p-nitrophenyl-β-D-galactopyranoside (PNP), 4-methylumbelliferyl-β-D-galactopyranoside and 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-GAL). However, it is clear that β-galactosidase II is also active against natural substrates, i.e., β(1→4)galactan (Carey et al., 1995; Carrington and Pressey, 1996; Pressey, 1983). β-Galactosidase proteins have been purified and characterized in a number of other fruits including kiwifruits (Ross et al., 1993), coffee (Golden et al., 1993), persimmon (Kang et al., 1994), and apple (Ross et al., 1994).
Carey et al. (1995) were able to purify three previously identified β-galactosidases from ripening tomato fruit (Pressey, 1983), but only one (β-galactosidase II) was active against β(1→4)galactan. Even though they were able to identify putative β-galactosidase cDNA clones, none of the cDNA's deduced amino acid sequences matched the amino terminal sequence of the β-galactosidase II protein. Although β-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan has been purified, cloning of the corresponding gene has been elusive.
The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial (truncated) sense RNA has been utilized to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic-Engineering Reviews 9:207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al, 1992, Plant Molecular Biology, i9:69-87). Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to “over-express” the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences. The complete disclosure of each of the references cited above is fully incorporated herein by reference.
The need therefore exists to clone a gene for β-galactosidase II and related polypeptides, and using known methods of modification of plant gene expression, thereby to provide methods for modifying quality of fruits, particularly by modifying the cell wall, thereby directly affecting the ripening of the fruit;